Alzheimer's disease (AD) is an age-dependent neurodegenerative disorder that leads to profound behavioral changes and dementia. Hallmark pathologies include the atrophy of brain grey matter as a result of the massive loss of neurons and synapses, and protein deposition in the form of both intraneuronal neurofibrillary tangles and extracellular amyloid plaques within the brain parenchyma. In addition, affected areas of the AD brain exhibit a reactive gliosis that appears to be a response to brain injury. Surviving neurons from vulnerable populations in AD show signs of metabolic compromise as indicated by alterations in the cytoskeleton (Wang et al., Nature Med. 2: 871-875 (1996)), Golgi complex (Salehi et al., J. Neuropath. Exp. Neurol. 54: 704-709 (1995)) and the endosomal-lysosomal system (Cataldo et al., Neuron 14: 671-680 (1995)).
Approximately 10 to 30% of AD cases are inherited in an autosomal dominant fashion and are referred to as "familial Alzheimer's disease" or "FAD." Genetic linkage studies have revealed that FAD is heterogeneous and a majority of the cases have been linked to gene mutations on chromosomes 1, 14, 19, or 21 (reviewed in Siman and Scott, Curr. Opin. Biotech. 7: 601-607 (1996)). Importantly, these individuals have been shown to develop the classical symptomatological and pathological profiles of the disease confirming that the mutations are associated with the development of the disease rather than a related syndrome. The locus on chromosome 14 is associated with a significant fraction of FAD, and mutations at the locus have been mapped to a single-copy gene, termed "S182" or "presenilin 1" (PS-1), that encodes a 467 amino acid protein (Sherrington et al., Nature 375: 754-760 (1995); Clark et al. Nature Genet. 11: 219-222 (1995)). A closely related gene, "STM2" or "presenilin 2" (PS-2), located on chromosome 1, has been linked to two additional FAD kindreds including the descendants of German families from the Volga valley of Russia (Levy-Lahad et al., Science 269: 973-977 (1995); Rogaev et al., Nature 376: 775-778 (1995)). PS-1 and PS-2 share an overall 67% amino acid sequence homology, and primary structure analysis indicates they are integral membrane proteins with 6 to 8 trans-membrane domains (Slunt et al., Amyloid--Int. J Exp. Clin. Invest. 2: 188-190 (1995); Doan et al., Neuron 17: 1023-1030 (1996)). Much of the information on function of the presenilins stems from the identification of a presenilin homolog in C. elegans termed "SEL-12", a 6 to 8 trans-membrane protein that appears to participate in an intracellular signaling pathway mediated by the lin-12/glp-1/Notch family (Levitan and Greenwald, Nature 377: 351-354 (1995)). PS-1 and SEL-12 proteins share a 48% sequence homology and have similar membrane orientations. Importantly, both human PS-1 and PS-2 can completely rescue the mutant sel-12 phenotype in C. elegans, indicating a role for the presenilins in Notch signaling (Levitan et al., Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)).
FAD linked to the presenilins is highly penetrant and the aggressive nature of the disease suggests that the mutant protein participates in a seminal pathway of AD pathology. To date, over thirty five FAD mutations have been identified in PS-1, and two FAD mutations have been found in PS-2. All of the FAD mutations occur in conserved positions between the two presenilin proteins, suggesting that they are affecting functionally or structurally important amino acid residues. Interestingly, many of the mutated amino acids are also conserved in SEL-12. All but one of the presenilin mutations are missense mutations and the exception results in an aberrant RNA splicing event that eliminates exon 10, creating an internally-deleted mutant protein (reviewed in Haas, Neuron 18: 687-690 (1997)). These latter points, along with the genetic dominance of the disease, argue that disease pathogenesis in the presenilin kindreds requires the production of a mutant presenilin protein having a novel and detrimental function, rather than the simple loss or reduction of normal presenilin levels. The mutations do appear to disrupt normal presenilin function however, because mutant presenilins are not able to rescue or fully rescue the sel-12 phenotype (Levitan et al., Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)).
Expression profiles of the presenilins have been examined at a gross level but, so far, these analyses have yielded little information on the mechanism of disease pathogenesis. Both presenilin 1 and 2 are widely expressed in the CNS and peripheral tissues. In brain, expression is enriched in neurons but is apparent in both AD-vulnerable and resistant areas. Cellular localization studies indicate that the proteins accumulate primarily in the Golgi complex and endoplasmic reticulum but no significant alterations in expression levels or subcellular distribution have been attributed to the FAD mutations (Kovacs et al., Nature Med. 2: 224-229 (1996)).
The presenilin proteins are processed proteolytically through two intracellular pathways. Under normal conditions, accumulation of 30 kD N-terminal and 20 kD C-terminal proteolytic fragments occurs in absence of the full-length protein. This processing pathway is highly regulated and appears to be relatively slow, accounting for turnover of only a minor fraction of the full-length protein. The remaining fraction appears to be rapidly degraded in a second pathway by the proteasome (Thinakaran et al., Neuron 17: 181-190 (1996); Kim et al., J. Biol. Chem. 272: 11006-11010 (1997)). Proteolytic metabolism of PS-1 variants linked to FAD appears to be different, but the relevance of the change to pathogenesis is not known (Lee, et al., Nature Med 3: 756-760 (1997)).
One pathogenic role for the mutant presenilins in FAD appears to be related to effects on processing of the amyloid precursor protein (APP) and production of the A.beta. peptide, the primary proteinaceous component of the extracellular neuritic plaque in the AD brain. Elevated serum levels of the longer form of A.beta. (A.beta.42), considered to be the more pathogenic species of the A.beta. peptides, have been measured in patients bearing PS-1 and PS-2 mutations (Scheuner et al., Nature Med. 2: 864-870 (1996)). Furthermore, overexpression of mutant, but not wild-type, presenilins in cell culture or transgenic mice results in enhanced secretion or production of A.beta.42 relative to A.beta.40 (Borchelt et al., Neuron 17: 1005-1013 (1996)). The mechanism by which the mutant presenilins affect APP processing is not known, but these results do support a causative role of increased A.beta.42 production in the development of FAD. Importantly, it is possible that mutant presenilins influence other AD pathogenic processes as well, such as presumptive intracellular signaling and cell death pathways involved directly or indirectly in neuronal dysfunction and degeneration.
Genetically-engineered animals have been used extensively to examine the function of specific gene products in vivo and their role in the development of disease phenotypes. The genetic engineering of mice can be accomplished according to at least two distinct approaches: (1) a transgenic approach where an exogenous gene is randomly inserted into the host genome, and (2) a gene-targeting approach where a specific endogenous DNA sequence or gene is partially or completely removed or replaced by homologous recombination. The transgene of a transgenic organism is comprised generally of a DNA sequence encoding the protein sequence and a promoter that directs expression of the protein coding sequences. A transgenic organism expresses the transgene in addition to all normally-expressed native genes. The targeted gene of a gene-targeted animal, on the other hand, can be modified in one of two ways: (1) a functional form where a modified version of the targeted gene is expressed, or (2) a non-functional or "null` form where the targeted gene has been disrupted resulting in loss or reduction of expression. If the targeted gene is a single copy gene and the animal is homozygous at the targeted locus, then, depending on the type of modification, the animal either does not express the targeted gene or expresses only a modified version of the targeted gene in absence of the normal form.
Transgenic mice expressing native and mutant forms of the presenilin proteins have been described (Borchelt et al., Neuron 17: 1005-1013 (1996); Duffet al., Nature 383: 710-713)). To date, no neuropathological features or behavioral deficits associated with AD have been described in these animals. However, selective elevations in brain levels of A.beta.42 have been found in animals expressing mutant, but not wild-type, PS-1. Gene-targeted PS-1 null mice lacking one or both functional alleles of the PS-1 gene have also been described (Wong et al., Nature 387: 288-292 (1997)). Mice in which both PS-1 alleles have been disrupted resulting in the complete loss of PS-1 expression are not viable and die shortly after birth. No abnormal phenotypes or changes in APP processing have been reported in mice lacking only one of the two PS-1 alleles.